This invention relates to biopolymer arrays, particularly polynucleotide arrays such as DNA arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
Arrays of biopolymers, such as arrays of peptides or polynucleotides (such as DNA or RNA), are known and are used, for example, as diagnostic or screening tools. Such arrays include regions (sometimes referenced as spots) of usually different sequence biopolymers arranged in a predetermined configuration on a substrate. The arrays, when exposed to a sample, will exhibit a pattern of binding which is indicative of the presence and/or concentration of one or more components of the sample, such as an antigen in the case of a peptide array or a polynucleotide of particular sequence in the case of a polynucleotide array. The binding pattern can be detected, for example, by labeling all potential targets (for example, DNA) in the sample with a suitable label (such as a fluorescent compound), and accurately observing the fluorescence pattern on the array.
Biopolymer arrays can be fabricated using in situ synthesis methods or deposition of the previously obtained biopolymers. The in situ synthesis methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 and the references cited therein for synthesizing polynucleotides (specifically, DNA). Such in situ synthesis methods can be basically regarded as repeating at each spot the sequence of: (a) deprotecting any previously deposited monomer so that it can now link with a subsequently deposited protected monomer; and (b) depositing a droplet of another protected monomer for linking. Different monomers may be deposited at different regions on the substrate during any one iteration so that the different regions of the completed array will have different desired biopolymer sequences. One or more intermediate further steps may be required in each iteration, such as oxidation, capping and washing steps. The deposition methods basically involve depositing biopolymers at predetermined locations on a substrate which are suitably activated such that the biopolymers can link thereto. Biopolymers of different sequence may be deposited at different regions of the substrate to yield the completed array. Washing or other additional steps may also be used. Reagents used in typical in situ synthesis are water sensitive, and thus the presence of moisture should be eliminated or at least minimized.
Typical procedures known in the art for deposition DNA such as whole oligomers or cDNA, are to load a small volume of DNA in solution on the tip of a pin or in an open capillary and touch the pin or capillary to the surface of the substrate. When the fluid touches the surface, some of the fluid is transferred. The pin or capillary must be washed prior to picking up the next type of DNA for spotting onto the array. This process is repeated for many different sequences and, eventually, the desired array is formed. Alternatively, the DNA can be loaded into an inkjet head and fired onto the substrate. Such a technique has been described, for example, in PCT publications WO 95/25116 and WO 98141531, and elsewhere. This method has the advantage of non-contact deposition. Still other methods include pipetting and positive displacement pumps such as the Bio-Dot A/D3000 Dispenser available from Bio-Dot Inc., Irvine, Calif., USA. There are four important design aspects required to fabricate an array of bioplymers such as cDNA""s or DNA oligomers. First, the array sensitivity is dependent on having reproducible spots on the substrate. The location of each type of spot must be known and the spotted area should be uniformly coated with the DNA. Second, since DNA is expensive to produce, a minimum amount of the DNA solution should be loaded into any of the transfer mechanisms. Third, any cross contamination of different DNA""s must be lower than the sensitivity of the final array as used in a particular assay, to prevent false positive signals. Therefore, the transfer device must be easily cleaned after each type of DNA is deposited or the device must be inexpensive enough to be a disposable. Finally, since the quantity of the assay sample is often limited, it is advantageous to make the spots small and closely spaced.
Similar technologies can be used for in-situ synthesis of biopolymer arrays, such as DNA oligomer arrays, on a solid substrate. In this case, each oligomer is formed nucleotide by nucleotide directly in the desired location on the substrate surface. This process demands repeatable drop size and accurate placement on the substrate. It is advantageous to have an easily cleaned deposition system since some of the reagents have a limited lifetime and must be purged from the system frequently. Since reagents, such as those used in conventional phosphoramidite DNA chemistry may be water sensitive, there is an additional limitation that these chemical reagents do not come in contact with water or water vapor. Therefore, the system must isolate the reagents from any air that may contain water vapor for hours to days during array fabrication. Additionally, the materials selected to construct system must be compatible with the chemical reagents thereby eliminating a lot of organic materials such as rubber.
Given the above requirements of biopolymer array fabrication, deposition using an inkjet type head is particularly favorable. In particular, inkjet deposition has advantages which include producing very small spot sizes. This allows high-density arrays to be fabricated. Furthermore, the spot size is uniform and reproducible as demonstrated by the successful use of inkjets in printers. Since it is a non-contact technique, ink-jet deposition will not scratch or damage the surface. Ink-jets have very high deposition rate, which facilitates rapid manufacture of arrays.
However, an ink-jet deposition system used for fabricating a biopolymer array, should meet a number of requirements. Specifically, the inkjet head must be capable of being loaded with very small volumes of DNA solution and function with minimal or no priming of the inkjets. The system should provide for easy purging of the working solution and readily flushed clean when required. When used for in-situ synthesis, the system should be able to keep reagents isolated from moisture in the surrounding air. Additionally, use of an inkjet head typically requires that a negative backpressure (that is, a pressure behind an orifice of the jet), in the range of one to six inches of water, be supplied to the inkjet head so that the inkjets form repeatable droplets without dripping during times when the jet has not been activated.
Open-cell foam has been used to provide this negative backpressure in an inkjet printer in a manner disclosed in U.S. Pat. No. 4,771,295, such the capillarity of the foam creates the negative backpressure in an ink reservoir. While this is an easy and economical way to provide the required negative backpressure, the foam cannot be easily purged of the working fluid. A small rubber thimble, similar to an eyedropper, can alternatively be used but the backpressure will vary as the reservoir is depleted. In addition, rubber is incompatible with the chemical reagents typically used in in-situ synthesis. A spring bag reservoir can be designed to control the backpressure of the fluid reservoir, however it requires a large working volume and is therefore not a good choice for the small reservoir volumes required by DNA or other biopolymer array fabrication. A regulated vacuum source could also be used. However, this may permit undesirable components, such as moisture, entering the head particularly during in situ synthesis. Additionally, purging the inkjet head then requires extra valves and a compressed nitrogen (or other suitable gas) source. Gravity is one of the easiest backpressure control means, however the backpressure changes as the fluid height drops and it requires too large a fluid volume to work properly for the small volumes encountered in an inkjet. It would be desirable then, to provide an apparatus and method for fabricating arrays of biopolymers which can use an inkjet type head or other pulse jet head, and which provides for easy purging and cleaning of the head. It would further be desirable that such an apparatus and method provide a simple way of providing a controlled negative backpressure to the head and also provide a simple way of purging the head, without an overly complex system of valves. It would also be desirable that any apparatus and method facilitates isolating reagents in the head from moisture or other undesirable components, and that it is of a compact construction given the small size of other components typically encountered in polynucleotide synthesis.
The present invention then, provides a method of fabricating an array of different or the same moieties (for example, multiple different chemical compounds) on a substrate using one or more suitable fluids, and using a fluid dispensing head. The invention is particularly useful for the in situ process since it provides the required head pressure while facilitating isolation of reagents from moisture or other undesirable components. However, the invention is also applicable to the direct deposition of polynucleotides. Particularly, the invention provides a method of fabricating an array of biopolymers using a biopolymer containing fluid, or one or more fluids containing a biomonomer. The head has at least one jet which can dispense droplets of a fluid onto a substrate, the jet including a chamber with an orifice, and including an ejector which, when activated, causes a droplet to be ejected from the orifice. The head may particularly be of a type commonly used in inkjet printers, in which a plurality of pulse jets (such as those with thermal or piezoelectric ejectors) are used, with their orifices on a common front surface of the head.
The method comprises positioning the head with the orifice facing the substrate. Multiple fluid droplets of the biopolymer, biomonomer or other fluid, are dispensed from the head orifice so as to form an array of droplets on the substrate (this formed array may or may not be the same as the final desired array since, for example, multiple heads can be used to form the final array). A gas flow is directed through a venturi which has a throat opening communicating with the dispensing head chamber. The gas used may be any suitable gas which may be selected depending upon the reagent chemistry. For example, when phosphoramidite oligonucleotide synthesis or other water sensitive chemistries are used, the gas should preferably be an inert anhydrous compressed gas such as anhydrous nitrogen. By xe2x80x9cinertxe2x80x9d in this context is referenced no substantial adverse reaction with a reagent. Gas flow rate through the venturi may be adjusted to alter the chamber pressure. This adjustment can occur whenever it is desired to change the pressure in the chamber, for example before or after the dispensing step. The adjustment can be accomplished by suitable means such as a valve on the venturi inlet and/or outlet side, or some other way of at least partially obstructing the inlet and/or outlet side (for example, an operator may simply manually block the outlet side). It will be appreciated from this arrangement, that all of the pressures in or at various chambers in the head therefore, are typically gas pressure (that is, provided by a gas in the location specified).
The venturi throat opening may provide a negative spotting pressure to the head chamber during dispensing of the droplets, and the gas flow resistance of the venturi outlet side may be adjusted (before or after dispensing) to provide a positive chamber pressure. This positive pressure may be provided by increasing the gas flow resistance of the venturi outlet side before dispensing (for example, as a priming pressure so as to assist in priming the jets) or after dispensing (for example, as a purging pressure so as to assist in purging any fluid remaining in the chamber out through the orifice). The priming and purging pressures may be the same or different, and each will typically be higher than the spotting pressure. In the case of purging, a purge fluid may optionally be added to the head chamber prior to providing the purging pressure.
In one aspect of the method, which is particularly useful for (but not limited to) the in situ method, the chamber is loaded with the fluid from a direction behind the orifice (that is, liquid is not loaded through the orifice). Following loading, the gas flow resistance of the venturi outlet side is increased to provide a positive priming pressure to the chamber. This assists in forcing liquid into the one or more jets to prime them.
In another aspect, which is particularly useful for (but not limited to) the deposition of previously obtained biopolymers, the method may additionally include, prior to the dispensing step, loading the head with a fluid, such as a fluid containing a biomonomer (for example, a nucleotide reagent), biopolymer (for example, a pre-synthesized oligonucleotide, cDNA, or DNA purified or amplified from a natural source), or other fluid (for example a fluid containing a moiety or a reagent used in producing such chemical a moiety). This loading step includes positioning the head facing a load station which is spaced from the substrate, with the one or more orifices adjacent and facing the fluid to be loaded. A loading pressure is provided in the chamber from the venturi throat opening while the head is facing the load station, which is sufficiently negative such that the fluid is drawn into the chamber through the one or more orifices. The gas flow rate through the venturi is adjusted to provide a spotting pressure to the chamber while dispensing droplets from the head, which spotting pressure may be the same or higher (that is, less negative) than the loading pressure. This adjustment may, for example, be accomplished by adjusting a valve on the inlet side of the venturi.
The method may include the loading, spotting and purging steps as described above.
In another aspect, the present invention provides a method of fabricating an array of different moieties, particularly biopolymers on a substrate using a biopolymer or biomonomer fluid, and using a fluid dispensing head as described above, which method includes positioning the head with the orifice facing the substrate. Multiple fluid droplets of the biopolymer, biomonomer, or other fluid are dispensed from the head so as to form an array of droplets on the substrate. A flow of inert anhydrous gas is directed through a venturi which has a throat opening communicating with the dispensing head chamber. This aspect may additionally include providing any of the loading, spotting and purging pressures, in the same manners as mentioned above. The head used in the method may have multiple pulse jets with orifices on a common front face of the head, such as a typical inkjet printing head. In this case, some or all of the jets can be loaded with the same or different fluids (biopolymer or otherwise, for example, deprotection reagent or other reagent).
An apparatus which can be used to execute a method of the present invention is also provided. In one aspect, the apparatus comprises a substrate station on which the substrate can be mounted, and a fluid dispensing head, and venturi, all as described above. The apparatus may further include a source of inert anhydrous gas communicating with the venturi pressurized inlet, and/or a valve to adjust the gas flow rate through the venturi (the valve being on the inlet or outlet side of the venturi, or a valve can be provided on both sides). A positioning system moves at least one of the dispensing head and mounted substrate with respect to the other, so that multiple droplets dispensed from the head onto the substrate will form an array thereon. The apparatus may further include, particularly in the aspect used for deposition of previously obtained biopolymers, the load and purge stations. A control processor may be present to operate the positioning system to selectively position the head facing any one of the stations, and which processor also adjusts the venturi outlet control valve to any of the required positions. In a particular embodiment, the load station comprises a plate on which multiple drops of different solutions can be retained.
The method and apparatus of the present invention can provide a simple way of controlling backpressure in a pulse type fluid dispensing head, and can also provide a simple way of purging the head, without requiring an overly complex system of valves. The apparatus and method can also facilitate isolating reagents in the head from moisture or other undesirable components.